1. Field of the Invention
The present invention relates to the field of biological assays and apparatus for carrying out such assays, such as a microfluidic device, to which is applied electric fields to control movement of charged molecules. The assays involve charged molecular species, such as nucleotides (due to phosphate ions), or other molecules which contain a charge due to their ionic nature, such as certain proteins or small molecules.
2. Related Art
Advances in silicon microfabrication have been used to produce microchannels and microarrays for many lab-on-a-chip platforms. Advantages include low reagent costs, miniaturization, and fast reaction rates. However, the challenge is to efficiently isolate and deposit biological samples into individual wells for high-throughput analysis. Recently, random arrays have been implemented in which solid-supports are used to individually capture unique biological molecules and deposit these solid supports into reaction wells with a geometry of the same size range. Another challenge these platforms are faced with is when repetitive assay are performed on the same bead isolated within a well. A good example where these challenges are common is DNA sequencing.
In certain methods of DNA sequencing, DNA is immobilized on a solid support, and nucleotides and enzymes are delivered to the DNA for successive incorporation of nucleotides. This is commonly referred to as DNA sequencing using sequencing-by-synthesis. Nucleotides are removed through washing to allow iterative nucleotide addition. One of the main challenges in sequencing by synthesis is to deliver the nucleotide to the vicinity of DNA to enable rapid incorporation and to remove the nucleotide efficiently to enhance the read-length.
Particular Patents and Publications
Dressman et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations,” Proc Nat Acad Sci Jul. 22, 2003, vol. 100, no. 15, pp 8817-8822, discloses a technique in which each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). After PCR cycling, the microemulsion is broken by detergent, and the beads are separated from the oil phase by centrifugation, and by placing the tube on an MPC-S magnet from Dynal.
Margulies et al., “Genome sequencing in microfabricated high-density picolitre reactors,” Nature 437, 376-380 (2005) discloses a method and apparatus for sequencing by synthesis which uses open wells of a fiber optic slide. The method uses a modified pyrosequencing protocol that is designed to take advantage of the small scale of the wells. The fiber optic slides are manufactured by slicing of a fiber optic block that is obtained by repeated drawing and fusing of optic fibers. The slide, containing approximately 1.6 million wells, is loaded with beads and mounted in a flow chamber designed to create a 300-mm high channel, above the well openings, through which the sequencing reagents flow. The unetched base of the slide is in optical contact with a second fiber optic imaging bundle bonded to a charge-coupled device (CCD) sensor, allowing the capture of emitted photons from the bottom of each individual well. 800 ml of emulsion containing 1.5 million beads are prepared in a standard 2-ml tube. Each emulsion is aliquotted into eight PCR tubes for amplification. After PCR, the emulsion is broken to release the beads, which include beads with amplified, immobilized DNA template and empty beads.
The enriched template-carrying beads are deposited by centrifugation into open wells. Streptavidin-coated SeraMag beads are bound to the biotinylated enrichment primers annealed to the immobilized templates on the DNA capture beads. It is essential not to vortex the beads, as vortexing may break the link between the SeraMag and DNA capture beads.
Erickson et al., “Electrokinetically Based Approach for Single-Nucleotide Polymorphism Discrimination Using a Microfluidic Device,” Anal. Chem., 77 (13), 4000-4007, (2005) discloses an electrokinetic approach for single-nucleotide polymorphism (SNP) discrimination using a PDMS/glass-based microfluidic chip. The technique takes advantage of precise control of the coupled thermal (Joule heating), shear (electroosmosis), and electrical (electrophoresis) energies present at an array of probes afforded by the application of external electrical potentials. A four-port device is described, with different voltages applied to different ports.
Chen et al., “Nanopore sequencing of polynucleotides assisted by a rotating electric field,” Applied Physics Letters volume 82, number 8, 24 Feb. 2003 1308-1310 disclose a method to control the translocation processes of polynucleotides through a nanopore assisted by a rotating electric field. Although the work is based on a simulation, it is stated that the method can be easily implemented in a nanopore sequencing experiment by adding two pairs of parallel electrodes above the thin film.
Erickson, D., Liu, X., Krull, D., Li, D. “An electrokinetically controlled DNA hybridization microfluidic chip enabling rapid target analysis,” Analytical Chemistry, 2004, 76, 7269-7277, discloses a device in which different voltages are applied to different ends of an “H” shaped flow channel. The paper further describes chip fabrication techniques.
Edman et al., “Electric field directed nucleic acid hybridization on microchips,” Nucleic Acids Research, Vol 25, Issue 24 4907-4914, discloses a microchip-based nucleic acid array where electronic addressing and/or hybridization is carried out by selective application of a DC positive bias to the individual microelectrodes beneath the selected test sites. This causes rapid transport and concentration of negatively charged nucleic acid molecules over selected locations on the microelectronic array. The nucleic acid (DNA, RNA, polynucleotides, oligonucleotides, etc.) may then be immobilized by direct attachment to the permeation layer overlying the microelectrode or by hybridization to previously addressed and attached nucleic acids. This paper describes buffer conditions and the like which may be adapted in practicing the methods taught here. Sosnowski, R. G., Tu, E., Butler, W. F., O'Connell, J. P. and Heller, M. J. Proc. Natl. Acad. Sci. USA, 1997, 94, 1119-1123 (cited in this paper) demonstrates that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength.
Horejsh et al., “A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry,” Nucleic Acids Res., 2005, 33(2): e13. discloses another assay format using beads. In this case, a fluid array system using microsphere-conjugated molecular beacons uses a flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons are conjugated with microspheres using a biotin-streptavidin linkage.
U.S. Pat. No. 6,287,774 to Nikiforov, issued Sep. 11, 2001, entitled “Assay methods and system,” discloses an assay system comprising a first channel disposed in a body structure. The first channel is fluidly connected to a source of a first reagent mixture, which comprises a first reagent having a fluorescent label, a source of a second reagent that reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent; and a source of a polyion. The system also includes a material transport system for introducing the first reagent, the second reagent and the polyion into the first channel and a detector disposed in sensory communication with the first channel. The detector is configured to detect the level of fluorescence polarization of reagents in the detection zone.
As referenced in the above patent, a controlled electrokinetic transport system is described in detail in U.S. Pat. No. 5,858,195, to Ramsey. Such electrokinetic material transport and direction systems include those systems that rely upon the electrophoretic mobility of charged species within the electric field applied to the structure. Such systems are more particularly referred to as electrophoretic material transport systems. Other electrokinetic material direction and transport systems rely upon the electroosmotic flow of fluid and material within a channel or chamber structure, which results from the application of an electric field across such structures. In brief, when a fluid is placed into a channel, which has a surface bearing charged functional groups, e.g., hydroxyl groups in etched glass channels or glass microcapillaries, those groups can ionize. In the case of hydroxyl functional groups, this ionization, e.g., at neutral pH, results in the release of protons from the surface and into the fluid, creating a concentration of protons at near the fluid/surface interface, or a positively charged sheath surrounding the bulk fluid in the channel. Application of a voltage gradient across the length of the channel, will cause the proton sheath to move in the direction of the voltage drop, i.e., toward the negative electrode.
U.S. Pat. No. 6,733,244 to Fritsch, et al., issued May 11, 2004, entitled “Microfluidics and small volume mixing based on redox magnetohydrodynamics methods,” discloses a device where microfluidic channels utilizing magnetohydrodynamics are used to pump very small volumes of solution. The channels have electrodes along the walls of the channel and a current carrying species within the solution carries the current through the solution. The electric field generated by the use of the current carrying species is perpendicular to a magnetic field applied to the channel. The two fields are applied perpendicular to the desired direction of flow. The combination of the electric and magnetic fields causes the solution to flow through the channel, perpendicular to both fields.
It should be noted that the present devices provide an electric field, which can move charged particles (molecules) through a solution. The field does not move the solution itself. Furthermore, the field need not be electromagnetic, and does not rely on ferromagnetic principles to cause movement. That is, one here is not simply attracting beads with a magnet. This would not cause the particle movements described here.